| Influenza caused by influenza virus is an acute respiratory infectious disease, which seriously affects people’s health. Recently, influenza has already been a very sensitive topic. The constant reports of human infection of the highly pathogenic H5N1avain influenza virus, the global spreading of the novel H1N1influenza virus and the fist outbreak of human infection of H7N9in China have heightened public attentions to influenza. Neuraminidase (NA) is one major surface protein of type A and type B influenza virus. It can cleave the glycosidic bonds of terminal sialic acids from carbohydrates, glycolipids, or glycoproteins of target cells. In the life cycle of influenza virus, NA plays an important role in viral infection and replication, such as releasing virions away from infected cells, reducing the aggregation of new born viral particles, preventing viral inactivation and assisting the movement of virus through the mucus. So far, ten NA subtypes of type A influenza virus have been discovered. However, the NA active sites of type A and B influenza virus are highly conserved, which makes it to be an good target for the design of anti-influenza drugs. Since zanamivir (trade name Relenza) and oseltamivir (trade name Tamiflu) were approved in1999, NA inhibitors have been the main options for the prophylaxis and treatment of influenza, and also became a hot research topic in the field of anti-influenza drugs. Later, many other NA inhibitors with different scaffolds were also discovered, such as benzoic acid derivatives, cyclohexene derivatives, cyclopentane derivatives and polyvalent NA inhibitors. In several countries, another two potent NA inhibitors, peramivir (trade name Rapiacta) and laninamivir (trade name INAVIR) have already been approved.Among these marketed NA inhibitors, only oseltamivir is orally effective and has been widely used in many countries. However, in recent years oseltamivir-resistant viral strains are constantly emerging, like H5N1avain influenza viruses and some seasonal H1N1, H3N2influenza viruses, which seriously affects its clinically application. Zanamivir and peramivir are mainly administered by inhaled nebulized or intravenous routes. Though they are not frequently used, drug-resistant influenza viral strains are also found. Considering the current situation and the potential threat, it is still necessary to find novel effective drugs to cope with influenza virus.In the research, we made a deep exploration of influenza NA inhibitors by the means of ligand-based drug design and structure-based drug design, obtaining22classes of135compounds. Through the preliminary NA (H9N2and H5N1) inhibition screening, two classes of lead compounds were discovered. The dissertation is divided into six chapters, mainly containing three parts, which are as follows:Part1:It has been reported that many natural products, such as flavones, chalcones and some polyphenols exhibited good NA inhibitory activities and/or anti-influenza virus activities in cells. Their structures do not conform to the pharmacophore model of classical NA inhibitors, and are also greatly different from zanamivir and oseltamivir. In order to discover some novel NA inhibitors, in the present study, we first constructed one pharmaceutical model of some flavnoid NA inhibitors. Then, according to the model, we took2,4-diaminophenol as the basic fragments to build a compound library with structural diversity. Caffeic acid was found to be a weak NA inhibitor. We also made structural modifications of it to obtain three series of caffeic acid derivatives. Through in vitro NA inhibition assay and structural optimization, we obtained92compounds in total. Among them, caffeic acid derivatives exhibited the best NA inhibitory activities, and also presented anti-influenza virus activities in cells, such as compounds (G3),(G4),(M1),(M2) and (P2). In the kinetic studies, they were found to be non-competitive NA inhibitors which indicated that the binding patterns of them with NA were quite different from that of classical NA inhibitors. This kind of compounds was a new type of influenza NA inhibitors.Part2:In our previous studies, we discovered some NA inhibitors with medium activities originally from L-hydroxyproline (L-hyp). Based on this and the structural characters of five-membered ring NA inhibitors, we continued to make exploration of L-hyp derivatives as potential influenza NA inhibitors. Structural modifications of L-hyp derived three series of novel compounds:①converting the hydroxyl group at the C-4position into the guanidine group and introducing hydrophobic groups at the NH of the pyrrolidine ring;②converting the NH to an amidino group and the hydroxyl group at the C-4position into NH2that was acylated with hydrophobic acids;③converting the NH to an amidino group and the hydroxyl group at the C-4position into azides that was used to introduce triazole groups by Click reaction. On the whole, the three series of L-hyp derivatives presented weak NA inhibitory activities, and much less than the activity of oseltamivir carboxylate. Molecular docking analysis indicated that the groups of these L-hyp derivatives can’t bind properly with the NA active sites, which may lead to the poor activities.Part3:In2006, it was first reported that there was a large cavity, named150-cavity adjacent to the active site of group-1(N1, N4, N5, N8subtypes) but not in group-2(N2, N3, N6, N7, N9subtypes) NAs. This cavity was thought to be a great advantage for finding selective and potent NA inhibitors. In this part of our research, we took oseltamivir as the lead to design and synthesize two series of derivatives, the substituted guanidines (series T) and the secondary amines (Series U), by modifying the NH2at C-5position. We obtained31novel compounds in total, and their structures were confirmed by1H-NMR,13C-NMR and HRMS. By testing the inhibitory activities against N1and N2NAs, it was found that most of these compounds were N1selective, and the activities of series U were much better than that of series T.(U12) and (U19) were the best two compounds, which were about8-fold more potent than oseltamivir carboxylate against NA of the H5N1virus. Moreover, compound (U12) also showed excellent anti-influenza virus activity in chick embryo model. The docking analysis indicated the diphenylmethyl group of (U12) could occupy the150-cavity and the other structural part could bind with the enzyme’s active site, similar to the binding pattern of oseltamivir carboxylate. The result of our studies proved that the150-cavity can be utilized to develop potent and selective influenza NA inhibitors.Conclusion:In our studies, we mainly discovered two new kinds of influenza NA inhibitors, the caffeic acid derivatives and the oseltamivir derivatives by different strategies of rational drug design. Caffeic acid is one natural product that has a variety of potential pharmacological effects. The derivatives designed exhibit good activates against both of the enzyme and the virus. Several compounds are even better than most of the reported natural products. This kind of compounds is a new type of anti-influenza agents and deserve further studies. The highly pathogenic H5N1avain influenza virus and the H1N1influenza virus are a great threat to human. Since the150-cavity was reported, many studies had been conducted to explore N1selective NA inhibitors, but there were no brilliant progress in this aspect. In our studies, the highly potent and selective H5N1NA inhibitors are a breakthrough. We also provide one rational design strategy for discovery of Group-1specific NAs inhibitors. Under the current anti-influenza situation, our discovery is very meaningful and may provide a new research direction for the treatment of influenza. |
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